|Year : 2009 | Volume
| Issue : 2 | Page : 141-149
|Porphyromonas gingivalis decreases osteoblast proliferation through IL-6-RANKL/OPG and MMP-9/TIMPs pathways
Xuan Khanh Le, Claude Laflamme, Mahmoud Rouabhia
Oral Ecology Research Group, Faculty of Dentistry, Laval University, Québec City, Québec - G1K 7P4, Canada
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|Date of Submission||07-Aug-2007|
|Date of Decision||28-Sep-2007|
|Date of Acceptance||05-Oct-2007|
|Date of Web Publication||23-Jun-2009|
| Abstract|| |
Background: Porphyromonas gingivalis, an important periodontal pathogen, is closely associated with inflammatory alveolar bone resorption. This bacterium exerts its pathogenic effect indirectly through multiple virulence factors, such as lipopolysaccharides, fimbriae, and proteases. Another possible pathogenic path may be through a direct interaction with the host's soft and hard tissues (e.g., alveolar bone), which could lead to periodontitis.
Aims and Objectives: The aim of the present study was to investigate the direct effect of live and heat-inactivated P gingivalis on bone resorption, using an in vitro osteoblast culture model.
Results: Optical microscopy and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide MTT assay revealed that live P gingivalis induced osteoblast detachment and reduced their proliferation. This effect was specific to live bacteria and was dependent on their concentration. Live P gingivalis increased IL-6 mRNA expression and protein production and downregulated RANKL and OPG mRNA expression. The effect of live P gingivalis on bone resorption was strengthened by an increase in MMP-9 expression and its activity. This increase was accompanied by an increase in TIMP-1 and TIMP-2 mRNA expression and protein production by osteoblasts infected with live P gingivalis.
Conclusion: Overall, the results suggest that direct contact of P gingivalis with osteoblasts induces bone resorption through an inflammatory pathway that involves IL-6, RANKL/OPG, and MMP-9/TIMPs.
Keywords: Bone, IL-6, osteoblasts, osteoprotegerin, MMPs, Porphyromonas gingivalis, RANKL, TIMPs
|How to cite this article:|
Le XK, Laflamme C, Rouabhia M. Porphyromonas gingivalis decreases osteoblast proliferation through IL-6-RANKL/OPG and MMP-9/TIMPs pathways. Indian J Dent Res 2009;20:141-9
Periodontal diseases, which are chronic inflammatory disorders localized to the attachment structures of teeth, are considered to be the major cause of tooth loss in adults and the most prevalent form of bone pathology in humans. The destructive form of periodontal disease, periodontitis, is present at significant levels worldwide.  Periodontitis is characterized by the irreversible destruction of both soft and hard tissues. These effects appear to be the result of a complex interaction between the host and the oral microorganisms. Porphyromonas gingivalis ingivalis), a gram-negative, black-pigmented, strict anaerobic bacterium, is one of the major etiologic agents responsible for the initiation and progression of periodontitis. , Pathogenesis due to P gingivalis can result from indirect contact between the bacteria and the host tissue through various factors, including lipopolysaccharides (LPS), fimbriae, and cysteine proteinases. , These virulence factors are involved in tissue colonization and can alter the host's defences.  They diffuse into and infiltrate deeper periodontal tissue, destroying the infected tissue.  P gingivalis is a potent stimulator of inflammatory mediators such as interleukin-1 (IL-1) and prostaglandin E 2 , which eventually induce bone resorption. 
|How to cite this URL:|
Le XK, Laflamme C, Rouabhia M. Porphyromonas gingivalis decreases osteoblast proliferation through IL-6-RANKL/OPG and MMP-9/TIMPs pathways. Indian J Dent Res [serial online] 2009 [cited 2021 Apr 16];20:141-9. Available from: https://www.ijdr.in/text.asp?2009/20/2/141/52884
A second route P gingivalis may take to promote tissue destruction may be through direct interaction between bacteria and host cells. When in close contact with the epithelium in periodontal pockets, P gingivalis is capable of invading various host tissues, including the alveolar bone.  In periodontal disease alveolar bone loss is a key event. The integrity of bone tissue depends on a positive balance between bone regeneration and resorption.  This key physiological process involves osteoblasts through different mediators and their ligands. It is well established that macrophage colony-stimulating factor and receptor activator of nuclear factor-κB (RANK) and its ligand (RANKL) are important to osteoblast differentiation.  RANKL, RANK, and osteoprotegerin (OPG) play key roles in remodelling bone. RANKL modulates osteoclast differentiation and activation. OPG downregulates bone resorption through a RANK-RANKL pathway. , Imbalance in the RANKL-RANK/OPG ratio may be a pivotal component in the etiology of bone resorption diseases. These cellular mechanisms could be triggered when P gingivalis interacts directly with osteoblasts, thus leading to bone resorption and periodontitis exacerbation. The goal of the research described here was to investigate the effect of direct contact of live and heat-inactivated P gingivalis on bone resorption by studying the bacterium's effect on cell adhesion and proliferation, cytokine (IL-6) expression and secretion, and RANKL-RANK/OPG expression. We also investigated the effect of P gingivalis on proteases (MMP-2 and MMP-9) and protease inhibitors (TIMP-1 and TIMP-2) gene activation and protein production. The findings may provide some insight into the mechanisms by which host-bacterium interactions result in tissue loss in conditions such as periodontal disease.
| Materials and Methods|| |
MG-63 osteoblast cell cultures
The MG-63 human osteosarcoma cell line is widely used to study the biocompatibility of orthopedic and dental biomaterials. MG-63 osteoblast-like cells (American Type Culture Collection, Manassas, VA, USA) were subcultured in a 3:1 mixture of Dulbecco-Vogt's modified Eagle's (DME) medium and Ham's F-12 (H) (Invitrogen Life Technologies, Burlington, ON, Canada) supplemented with 24.3 µg/ml adenine, 10 µg/ml human epidermal growth factor (Chiron Corp., Emeryville, CA, USA), 0.4 µg/ml hydrocortisone (Calbiochem, La Jolla, CA, USA), 5 µg/ml bovine insulin, 5 µg/ml human transferrin, 2 × 10−9 M 3,3',5'-triiodo-L-thyronine, 10−10 M cholera toxin (Schwarz/Mann, Cleveland, OH, USA), 100 U/ml penicillin, 25 µg/ml gentamicin (Schering, Pointe-Claire, QC, Canada), and 10% fetal calf serum (NCS, fetal clone II; Hyclone, Logan, UT, USA). Subconfluent cell cultures were trypsinized and split 1:10 to maintain cell growth and avoid differentiation. The cell cultures were incubated at 37°C in a humid 5% CO 2 atmosphere.
P gingivalis growth and inactivation
P gingivalis (ATCC 33277, American Type Culture Collection, Manassas, VA, USA) was used throughout the study. Bacteria were routinely grown in Todd-Hewitt broth (THB) (BBL Microbiology Systems, Cockeysville, MD, USA) supplemented with haemin (10 µg/ml) and vitamin K (1 µg/ ml) and incubated for 48 h in an anaerobic chamber (N 2 /H 2 /CO 2 75:10:15) at 37°C. The bacteria were then harvested by centrifugation, washed three times in THB medium, and adjusted to 10 10 cfu/ml. To prepare inactive bacteria, the P gingivalis suspensions were autoclaved for 20 min at 120°C in a humid chamber. An aliquot of the heat-inactivated suspension was added to THB medium and incubated for 48 h at 37°C; this was examined after 24 h, and absence of bacterial growth confirmed inactivation.
Osteoblast culture in the presence of live and heat- inactivated P gingivalis
Osteoblasts were detached from the culture flasks using trypsin and were washed twice in culture medium. They were then counted, seeded into six-well tissue culture plates (Sarstedt Inc., Montrιal, QC, Canada) at 2.5 × 10 4 cells/well, and incubated at 37°C in an 8% CO 2 atmosphere. After 4-5 days of culture, the cells were cultured to approximately 80% confluence, then either infected or not infected with live and heat-inactivated P gingivalis (10 6 , 10 7 , 10 8 and 10 9 bacteria). The infected and noninfected osteoblasts were cultured for 24 and 48 h, respectively. Following each contact period, the cells were photographed and used to determine cell proliferation by means of a colorimetric assay.
Effect of P gingivalis on osteoblast adhesion and proliferation
Following infection of osteoblast culture with and without live and heat-inactivated P gingivalis, the cell adhesion was assessed by observation under an optical microscope. The proliferation of the P gingivalis-infected and noninfected osteoblasts was assessed using the 3- [4,5- dimethylthiazol- 2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay (Sigma, St. Louis, MO, USA), which measures cell growth as a function of mitochondrial activity.  It is based on the hydrolysis of the tetrazolium ring by mitochondrial dehydrogenase, which results in an insoluble blue reaction product (formazan). Briefly, a stock solution (5 mg/ml) of MTT was prepared inphosphate buffered saline (PBS) and added to each culture well at a final concentration of 1% (v/v). The P gingivalis-infected and noninfected osteoblast cultures were incubated with MTT for 4 h at 37°C. The supernatant was then removed and 2 ml of 0.04 N HCl in isopropanol was added to the culture wells, followed by an extended 15-min incubation. Finally, 200 µl of the reaction mixture (in triplicate) was transferred to the wells of a 96-well flat-bottom plate and the absorbance was measured at 550 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Model 680, BioRad Laboratories, Mississauga, ON, Canada). P gingivalis alone was also subjected to MTT assay, and the values obtained were deducted from the values obtained with infected osteoblasts. The results are reported as the means ± SD of eight separate experiments.
RT-PCR analysis of the effect of P gingivalis on IL-6, MMP, and TIMP gene activation
MG-63 cell cultures were inoculated with live and dead P gingivalis (10 7 bacteria), and cultured for 4, 8, and 24 h. Following each culture period, total cellular RNA was prepared from noninfected and live or heat-inactivated P gingivalis-infected osteoblast cultures using the Qiagen RNeasy Mini kit (Qiagen, Valencia, CA, USA). Concentrations of total RNA were measured by a fluorescent dye with RiboGreen RNA quantification reagent (Molecular Probes Inc., Eugene, OR, USA). RNA was reverse transcribed into cDNA using the Moloney murine leukemia virus (M-MLV) reverse transcriptase (Canadian Life Technologies, Burlington, ON, Canada) and random hexamers (Amersham Pharmacia Biotech, Oakville, ON, Canada). One microliter of each cDNA product was added to a 50-μl PCR mixture containing Taq polymerase (Qiagen) and forward and reverse primers (Medicorp, Inc., Montrιal, QC, Canada). Each reaction was performed in a Perkin-Elmer/Cetus DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT, USA). The cDNA was denatured by heating at 95°C for 2 min.
The PCR reactions were then performed under the following conditions: IL-6: 2 min at 95°C, 1 min at 94°C, 45 s at 56°C, and 1 min at 72°C for 35 cycles; MMP-2 and TIMP-2: 1 min at 95°C, 30 s at 56°C, and 1 min at 72°C for 35 cycles; MMP-9: 45 s at 95°C, 1 min at 60°C, and 1 min at 72°C for 35 cycles; TIMP-1:1 min at 95°C, 30 s at 56°C, and 1 min at 72°C for 30 cycles; RANK: 30 s at 94°C, 30 s at 68°C, and 1 min at 72°C for 40 cycles; RANKL: 30 s at 94°C, 30 s at 60°C, and 1 min at 72°C for 40 cycles; OPG: 30 s at 94°C, 30 s at 60°C, and 1 min at 72°C for 30 cycles; GAPDH: 1 min at 95°C, 30 s at 52°C, and 1 min at 72°C for over 35 cycles. After the final cycle, the temperature was maintained at 72°C for 10 min to enable further extension of the amplified products.
[Table 1] lists the primers used for the PCR. Following the PCR procedure, 8 μl of the products were separated on 2% agarose gel and observed after ethidium bromide staining. The relative intensity of the gel bands was measured using an image analyzing program (Scion Imaging Software, Scion Corp., Frederick, MD, USA). In order to exclude contaminating DNA from the isolated RNA, the RNA was subjected to PCR without cDNA synthesis. No bands were detected in any of the preparations following the PCR procedure.
IL-6, MMP-2, MMP-9, TIMP-1, and TIMP-2 secretion by osteoblasts following contact with live and heat- inactivated P gingivalis
The supernatants collected from the noninfected and P gingivalis-infected MG-63 cell cultures were used to determine the levels of secreted IL-6, MMP-2, MMP-9, TIMP-1, and TIMP-2. The tests were performed in triplicate using an ELISA (RandD Systems, Minneapolis, MN, USA) for these molecules. Supernatants were filtered through 0.22-µm filters and used to quantify the IL-6, MMP-2, MMP-9, TIMP-1, and TIMP-2 concentrations according to the manufacturer's instructions. The plates were read at 450 nm and analyzed using a Model 680 Microplate Reader (Biorad, Hercules, CA, USA). According to the manufacturer, the sensitivity of the ELISA kits was under 0.7 pg/ml for IL-6, 0.16 ng/ml for MMP-2, 0.156 ng/ml for MMP-9, 0.08 ng/ml for TIMP-1, and 0.011 ng/ml for TIMP-2. The experiments were repeated four times and the means ± SD were calculated and plotted.
Zymography of protease activity following osteoblast culture in the presence of live and heat- inactivated P gingivalis
Gelatin zymography was used to evaluate the activity of proenzymes and active MMP species produced by the infected cells. Culture supernatants were collected immediately following each contact period. Zymography was performed using the culture supernatants, as previously described.  These supernatants were diluted (9/10) in nonreducing sample buffer containing sodium dodecyl sulfate (SDS), glycerol, and bromophenol blue and were then subjected to electrophoresis on 10% polyacrylamide SDS gel containing 1 mg/ml porcine skin gelatin (Sigma). Following electrophoresis, the polyacrylamide gels were washed in 50 mM Tris-HCl (pH 7.4) and then in 2.5% Triton X-100/50 mM Tris-HCl (pH 7.4) to remove all traces of SDS. The gels were rinsed again in 50 mM Tris-HCl (pH 7.4) for 10 min and incubated overnight in digestion buffer containing 10 mM CaCl 2 and 100 mM NaCl. The gels were fixed in 10% methanol containing 10% acetic acid and stained with 0.05% Coomassie brilliant blue. Gelatin digestion was identified as clear zones of lysis against a blue background. The molecular weight of the gelatinolytic bands was estimated using SDS- PAGE protein standards ranging from 45 kDa to 200 kDa (Bio-Rad, Mississauga, ON, Canada).
Each experiment was performed at least three times. Experimental values are given as means ± SD. The statistical significance of the differences between the control and test values was evaluated using a one-way ANOVA t-test. The data were analyzed using SAS, version 8.2 (SAS Institute Inc., Cary, NC, USA).
| Results|| |
Downregulation of osteoblast adhesion and proliferation following direct contact with P gingivalis
A number of studies have shown that P gingivalis products such as LPS have side effects on mammalian cell adhesion and growth.  However, the direct effect of P gingivalis on mammalian cells, such as bone cell adhesion and proliferation, remains to be studied. In this context, we investigated the influence of live and heat-inactivated P gingivalis on osteoblast adhesion and growth. As shown in [Figure 1], observation under the microscope revealed that, compared to the noninfected cells, cultures infected with heat-inactivated P gingivalis showed no significant change in osteoblast adhesion and proliferation. In contrast, osteoblasts cultured in the presence of live P gingivalis displayed reduced cell density compared to noninfected osteoblast cultures and cultures infected with heat-inactivated P gingivalis. MTT analyses confirmed these observations. Indeed, the absorbance values were significantly lower (P < 0.01 to P < 0.05) in the osteoblast cultures infected with live P gingivalis than in the cultures infected with heat-inactivated P gingivalis and the noninfected cultures. Interestingly, as shown in [Figure 1], the concentration of live P gingivalis significantly affected osteoblast proliferation: the greater the bacterial concentration, the lower was the cell proliferation.
IL-6 expression and release by osteoblasts following contact with P gingivalis
As shown in [Figure 2], unlike the heat-inactivated microorganisms, live P gingivalis upregulated IL-6 expression. The effect of P gingivalis on IL-6 mRNA expression began as early as 4 h and was maintained until 8 h post-stimulation [Figure 2]. At 24 h post contact, the expression of the gene coding for IL-6 was not significantly different from that in the control. Compared to the peak IL-6 mRNA expression in the osteoblasts infected with heat-inactivated P gingivalis, the peak IL-6 mRNA expression in the live P gingivalis- infected osteoblasts occurred during the early contact period (4 h). It is interesting to note that only the live P gingivalis had a significant effect on the expression of the gene encoding for IL-6. The inactivated bacteria either induced no significant effect or caused a slight upregulation of IL-6 gene expression.
Observing these increased IL-6 mRNA levels in live P gingivalis-infected cells, we examined whether IL-6 protein was released into the culture supernatant and whether the release followed the same trend as mRNA production. The cytokine activity in the culture supernatants of osteoblasts exposed to the live bacteria was determined by ELISA and compared with constitutive IL-6 levels from the same experiments. In the absence of P gingivalis, osteoblasts secreted reduced amounts of IL-6 (15-20 ng/ml). However, when live P gingivalis were present, the levels of secreted IL-6 were significantly enhanced (P ≤ 0.01) [Figure 3]. IL-6 secretion was elevated at 4 and 8 h post contact, dropping to control levels after 24 h. Exposure to heat-inactivated P gingivalis did not promote IL-6 release, which suggests that only live bacteria are able to upregulate IL-6 secretion.
RANK, RANKL, and OPG expression by osteoblasts after contact with P gingivalis
[Figure 4] shows the expression levels of RANK, RANKL, and OPG. In the noninfected cultures, the osteoblasts expressed basal levels of these three molecules. These basal levels were significantly modulated following osteoblast infection with P gingivalis. Unlike the inactivated bacteria, live P gingivalis upregulated the expression of RANKL and OPG. The effect of P gingivalis on RANKL and OPG mRNA expression was not significant at 4 and 8 h post infection with either the heat-inactivated or the live P gingivalis. However, at 24 h post-infection, only those osteoblasts infected with live P gingivalis showed an important decrease in RANKL and OPG mRNA expression. At this later time point, RANK mRNA expression had slightly, although not significantly, increased in osteoblast cultures infected with live P gingivalis. Heat-inactivated P gingivalis therefore induced no effect (RANK and RANKL) or caused a slight upregulation (OPG) on the tested gene expression when compared to the noninfected osteoblast cultures.
P gingivalis increased MMP-9 mRNA expression
MMPs are particularly important in maintaining extracellular matrix (ECM) homeostasis. Any event that modifies the expression of these enzymes can disrupt the integrity of the ECM and allow microorganisms to invade deeper tissue.  We therefore investigated the effect of P gingivalis on MMP-2 and MMP-9 mRNA expression. Our results show that osteoblasts expressed higher basal levels of MMP-2 than of MMP-9 [Figure 5]. Following osteoblast infection with P gingivalis, the mRNA levels of MMP-2 were not modulated. Indeed, identical mRNA levels were found in the noninfected and the heat-inactivated and live P gingivalis-infected osteoblast cultures [Figure 5]. However, the bacteria induced an upregulation of the gene encoding for MMP-9. [Figure 5] shows that both the heat-inactivated and the live P gingivalis increased MMP-9 mRNA expression, as compared to the noninfected condition. Compared to the heat-inactivated P gingivalis, live P gingivalis significantly promoted the activation of the gene encoding for MMP-9.
MMP-9 proenzyme activation following P gingivalis infection
As P gingivalis was shown to have a greater impact on MMP-9 mRNA expression, the activity of secreted MMP-9 by the osteoblasts was evaluated by a gelatin zymogram using culture supernatants. As clearly illustrated in [Figure 6], a single 83 kDa band corresponding to the molecular mass of the active form of MMP-9 was produced. This lytic activity was present in the uninfected cells but was higher in the osteoblasts infected with P gingivalis; the MMP-9 band was more pronounced than was that of the control as early as 8 h post infection. At 24 h, no difference was noticed in the three conditions (noninfected and heat-inactivated and live P gingivalis-infected osteoblasts). Lytic bands obtained with supernatant collected from live P gingivalis-infected osteoblasts were significantly greater than those obtained with heat-inactivated P gingivalis. Interestingly, no lytic bands related to MMP-2 were observed in the zymography gels. These results suggest that direct contact of P gingivalis with osteoblasts contributes to the secretion of active MMP-9 by these cells.
P gingivalis modulation of TIMP-1 and TIMP-2 mRNA expression and protein production
MMP activity is extracellularly modulated by TIMPs which block the active site of MMPs.  These TIMPs may contribute to the modulation of the inflammatory response in various pathologies, including in microbial infections. In this context, we investigated the effect of P gingivalis on TIMPs. As shown in [Figure 7], the osteoblasts expressed basal levels of both TIMP-1 and TIMP-2. These inhibitors were increased by live P gingivalis but not by heat-inactivated P gingivalis. The only observable effect was obtained at 24 h after contact of the osteoblasts with P gingivalis, when the TIMP-1 and TIMP-2 levels increased significantly (P < 0.05) above the basal levels [Figure 7]. Of great interest is the nonactivation of the gene encoding for TIMPs by the heat-inactivated P gingivalis. In order to verify whether the effect of P gingivalis on TIMP mRNA expression was reflected in their secretion pattern, TIMP measurements (with an ELISA) were performed using culture supernatants collected from noninfected and P gingivalis-infected osteoblasts. As shown in [Figure 8], live P gingivalis highly modulated TIMP-1 and moderately modulated TIMP-2 production by the osteoblasts after 24 h of infection. The TIMP-1 and TIMP-2 levels obtained following live P gingivalis contact were both significantly high (P < 0.001 and P < 0.01, respectively), compared to the levels in the noninfected cultures and the heat-inactivated P gingivalis-infected cultures.
| Discussion|| |
Virulence factors released by periodontopathogens such as P gingivalis are involved in the chronic inflammation that leads to bone loss following infection. In association with inflammatory mediators, these virulence factors cause bone resorption through a pathway that requires the participation of osteoblasts.  The present study confirms this hypothesis by showing that following direct contact with the cells P gingivalis was able to reduce osteoblast adhesion and proliferation. These results are consistent with previous reports demonstrating that bacteria can induce the formation of osteoclastic cells without the formation of osteoblastic cells. ,, Our study and others in the literature suggest that in addition to an indirect effect (via released virulence factors), P gingivalis also has a direct effect on bone resorption through inhibition of osteoblast proliferation/growth.
Bone resorption is mediated by inflammatory cytokines, which may stimulate bone destruction by either enhancing the proliferation of osteoclasts or by promoting the differentiation or maturation of multinucleated cells to resorb bone or even both.  Inflammatory cytokines, such as IL-1 and TNF-a have been reported to indirectly induce osteoclast formation by stimulating osteoblasts. , IL-6 may also be involved in bone resorption following P gingivalis infection. Indeed, we showed that direct contact of live P gingivalis with osteoblasts promoted IL-6 mRNA expression and protein production. P gingivalis also stimulated RANKL/OPG gene activation, which may in turn promote osteoblast ingrowth/differentiation. On the other hand, the basal level of RANK expression by MG- 63 was not modulated by live or inactivated P gingivalis. This basal level expression of RANK by MG-63 supports the previous report by Mori et al., of RANK expression by human osteosarcoma cells.  .
These results are in agreement with those previously reported by Okahoshi et al., who showed that P gingivalis upregulates IL-6 mRNA expression in addition to RANKL.  Together, these data suggest that direct contact of P gingivalis with osteoblasts may promote osteoclastogenesis through the IL-6 and the RANKL/OPG pathway. Following inflammation, host cells produce multiple inflammatory cytokines and secrete various proteases, e.g., metalloproteinases. The over-expression of host cell-derived MMPs is linked to the pathological tissue destruction observed during periodontitis. , MMP activity is regulated through gene expression, conversion of the proenzyme to the active form, and the formation of complexes with TIMP-1 and TIMP-2.  MMP-2 and MMP-9, which are believed to play a key role in periodontal tissue destruction,  can be activated by bacterial components such as proteases. , In the present study, live P gingivalis was shown to significantly increase MMP-9 mRNA expression and its lytic activity. These results are in agreement with previous findings , showing that osteoblasts expressed more MMP-9 mRNA than MMP-2 following stimulation with P gingivalis extracts.
The effect of bacteria on protease release has also been documented with epithelial cells. When cultured in the presence of live P gingivalis, oral epithelial cells expressed high levels of MMP-9.  Through its effect on MMPs, P gingivalis affected the balance of MMPs/TIMPs by modulating the TIMP at the gene and protein levels. Recently, Zhou and Windsor (2006) demonstrated that P gingivalis increased the collagen-degrading ability of human gingival fibroblasts by increasing MMP activation while reducing the TIMP-1 protein level.  As TIMP-1 is an effective inhibitor of MMP-9,  the slight increase we observed in TIMP-1 mRNA expression and protein secretion following P gingivalis infection may have led to the higher MMP-9 levels and to the significant activity detected by RT-PCR and zymography.
The increased TIMP-1 expression and protein production reported in this study are the only results available on osteoblast cells infected with live P gingivalis. These findings are in accordance with a recent study  showing that the basal level of mRNA expression of TIMP-1 by human gingival fibroblasts increases following P gingivalis infection. Our study is the first to show that TIMP-2 expression and production by osteoblasts is modulated by live P gingivalis. Similar results have been reported with other cells but not with osteoblasts. Indeed, our research  and that of others  show that P gingivalis increases TIMP-2 expression and production by oral epithelial cells. This effect on MMP/TIMP balance is probably mediated by gingipains and other bacterial factors. A variety of bacterial components, including LPS and fimbriae, which are potent inducers of cytokine synthesis, may indirectly contribute to modulating MMP and TIMP expression. However, in the present study, both live and heat-inactivated P gingivalis were in direct contact with the osteoblasts, but only live bacteria were able to modulate protein expression; this suggests a significant effect of the bacteria on bone destruction through direct contact with the host hard tissue.
In conclusion, through direct contact, live P gingivalis reduced osteoblast growth via a possible IL-6- RANKL/ OPG- dependent pathway. P gingivalis was also able to affect the MMP/TIMP balance, leading to bone destruction through an MMP-9 pathway.
| Acknowledgments|| |
This study was financially supported by grants from the Natural Sciences and Engineering Research Council (discovery program), from the Fonds de la Recherche en Santι du Quιbec (FRSQ) and from the Fonds Émile-Beaulieu.
| References|| |
|1.||Papapanou PN. Epidemiology of periodontal diseases: An update. J Int Acad Periodontol 1999;1:110-6. [PUBMED] |
|2.||Lamont RJ, Jenkinson HF. Subgingival colonization by Porphyromonas gingivalis. Oral Microbiol Immunol 2000; 15:341-9. [PUBMED] [FULLTEXT]|
|3.||Holt SC, Kesavalu L, Walker S, Genco CA. Virulence factors of Porphyromonas gingivalis. Periodontol 2000 1999;20:168-238. |
|4.||Curtis MA, Aduse-Opoku J, Rangarajan M. Cysteine proteases of Porphyromonas gingivalis. Crit Rev Oral Biol Med 2001;12:192-216. |
|5.||Imamura T. The role of gingipains in the pathogenesis of periodontal diseases. J Periodontol 2003;74:111-8. [PUBMED] [FULLTEXT]|
|6.||Cutler CW, Kalmar JR, Genco CA. Pathogenic strategies of the oral anaerobe, Porphyromonas gingivalis. Trends Microbiol 1995;3:45-51. [PUBMED] [FULLTEXT]|
|7.||Noiri Y, Ozaki K, Nakae H, Matsuo T, Ebisu S. An immunohistochemical study on the localization of Porphyromonas gingivalis, Campylobacter rectus and Actinomyces viscosus in human periodontal pockets. J Periodontal Res 1997;32:598-607. [PUBMED] |
|8.||Hayashi S, Yamane T, Miyamoto A, Hemmi H, Tagaya H, Tanio Y, et al. Commitment and differentiation of stem cells to the osteoclast lineage. Biochem Cell Biol 1998;76:911-22. [PUBMED] [FULLTEXT]|
|9.||Yoshida H, Hayashi S, Kunisada T, Ogawa M, Nishikawa S, Okamura H, et al. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 1990;345:442-4. [PUBMED] [FULLTEXT]|
|10.||Chen T, Feng X. Cell-based assay strategy for identification of motif-specific RANK signaling pathway inhibitors. Assay Drug Dev Technol 2007;4:473-82. |
|11.||Wada T, Nakashima T, Hiroshi N, Penninger JM. RANKL-RANK signaling in osteoclastogenesis and bone disease. Trends Mol Med 2006;12:17-25. [PUBMED] [FULLTEXT]|
|12.||Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 1986;89:271-7. |
|13.||Rouabhia M, Deslauriers N. Production and characterization of an in vitro engineered human oral mucosa. Biochem Cell Biol 2002;80:189-95. [PUBMED] [FULLTEXT]|
|14.||Kadono H, Kido J, Kataoka M, Yamauchi N, Nagata T. Inhibition of osteoblastic cell differentiation by lipopolysaccharide extract from Porphyromonas gingivalis. Infect Immun 1999;67:2841-6. [PUBMED] [FULLTEXT]|
|15.||Nagase H, Woessner JF Jr. Matrix metalloproteinases. J Biol Chem 1999;274:21491-4. [PUBMED] [FULLTEXT]|
|16.||Tsuruda T, Costello-Boerrigter LC, Burnett JC Jr. Matrix metalloproteinases: Pathways of induction by bioactive molecules. Heart Fail Rev 2004; 9:53-61. [PUBMED] [FULLTEXT]|
|17.||Jiang Y, Mehta CK, Hsu TY, Alsulaimani FF. Bacteria induce osteoclastogenesis via an osteoblast-independent pathway. Infect Immun 2002;70:3143-8. [PUBMED] [FULLTEXT]|
|18.||Loomer PM, Ellen RP, Tenenbaum HC. Effects of Porphyromonas gingivalis 2561 extracts on osteogenic and osteoclastic cell function in co-culture. J Periodontol 1998;69:1263-70. [PUBMED] |
|19.||Mundy GR. Inflammatory mediators and the destruction of bone. J Periodontal Res 1991;26:213-7. [PUBMED] |
|20.||Charatcharoenwitthaya N, Khosla S, Atkinson EJ, McCready LK, Riggs BL. Effect of blockade of tumor necrosis factor-alpha and interleukin-1 action on bone resorption in early postmenopausal women. J Bone Miner Res 2007;22:724-9. [PUBMED] [FULLTEXT]|
|21.||Knudsen S, Harslof T, Husted LB, Carstens M, Stenkjaer L, Langdahl BL. The effect of interleukin-1alpha polymorphisms on bone mineral density and the risk of vertebral fractures. Calcif Tissue Int 2007;80:21-30. |
|22.||Mori K, Le Goff B, Berreur M, Riet A, Moreau A, Blanchard F, et al . Human osteosarcoma cells express functional receptor activator of nuclear factor-kappa B. J Pathol 2007;211:555-62. [PUBMED] [FULLTEXT]|
|23.||Okahashi N, Inaba H, Nakagawa I, Yamamura T, Kuboniwa M, Nakayama K, et al. Porphyromonas gingivalis induces receptor activator of NF-kappaB ligand expression in osteoblasts through the activator protein 1 pathway. Infect Immun 2004;72:1706-14. [PUBMED] [FULLTEXT]|
|24.||Birkedal-Hansen H. Role of matrix metalloproteinases in human periodontal diseases. J Periodontol 1993;64:474-84. [PUBMED] |
|25.||Sorsa T, Tjaderhane L, Salo T. Matrix metalloproteinases (MMPs) in oral diseases. Oral Dis 2004;10:311-8. |
|26.||Nagase H. Activation mechanisms of matrix metalloproteinases. Biol Chem 1997;378:151-60. [PUBMED] |
|27.||Makela M, Salo T, Uitto VJ, Larjava H. Matrix metalloproteinases (MMP-2 and MMP-9) of the oral cavity: Cellular origin and relationship to periodontal status. J Dent Res 1994;73:1397-406. |
|28.||Ding Y, Uitto VJ, Haapasalo M, Lounatmaa K, Konttinen YT, Salo T, et al Membrane components of Treponema denticola trigger proteinase release from human polymorphonuclear leukocytes. J Dent Res 1996;75:1986-93. |
|29.||Miyajima S, Akaike T, Matsumoto K, Okamoto T, Yoshitake J, Hayashida K, et al . Matrix metalloproteinases induction by pseudomonal virulence factors and inflammatory cytokines in vitro. Microb Pathog 2001;31:271-81. [PUBMED] [FULLTEXT]|
|30.||Chang YC, Chu SC, Yang SF, Hsieh YS, Yang LC, Huang FM. Examination of the signal transduction pathways leading to activation of gelatinolytic activity by interleukin-1a and Porphyromonas gingivalis in human osteosarcoma cells. J Periodontal Res 2004;39:168-74. [PUBMED] [FULLTEXT]|
|31.||Yun JH, Pang EK, Kim CS, Yoo YJ, Cho KS, Chai JK, et al. Inhibitory effects of green tea polyphenol (-)-epigallocatechin gallate on the expression of matrix metalloproteinase-9 and on the formation of osteoclasts. J Periodontal Res 2004;39:300-7. [PUBMED] [FULLTEXT]|
|32.||Andrian E, Mostefaoui Y, Rouabhia M, Grenier D. Regulation of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases by Porphyromonas gingivalis in an engineered human oral mucosa model. J Cell Physiol 2007;211:56-62. [PUBMED] [FULLTEXT]|
|33.||Zhou J, Windsor LJ. Porphyromonas gingivalis affects host collagen degradation by affecting expression, activation, and inhibition of matrix metalloproteinases. J Periodontal Res 2006;41:47-54. [PUBMED] [FULLTEXT]|
|34.||Strongin AY, Collier I, Bannikov G, Marmer BL, Grant GA, Goldberg GI. Mechanism of cell surface activation of 72-kDa type IV collagenase. Isolation of the activated form of the membrane metalloprotease. J Biol Chem 1995;270:5331-8. |
Oral Ecology Research Group, Faculty of Dentistry, Laval University, Québec City, Québec - G1K 7P4
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
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